Time-Resolved Imaging of High Mass Proteins and Metastable Fragments Using Matrix-Assisted Laser Desorption/Ionization, Axial Time-of-Flight Mass Spectrometry, and TPX3CAM

The Timepix (TPX) is a position- and time-sensitive pixelated charge detector that can be coupled with time-of-flight mass spectrometry (TOF MS) in combination with microchannel plates (MCPs) for the spatially and temporally resolved detection of biomolecules. Earlier generation TPX detectors used in previous studies were limited by a moderate time resolution (at best 10 ns) and single-stop detection for each pixel that hampered the detection of ions with high mass-to-charge (m/z) values at high pixel occupancies. In this study, we have coupled an MCP-phosphor screen-TPX3CAM detection assembly that contains a silicon-coated TPX3 chip to a matrix-assisted laser desorption/ionization (MALDI)-axial TOF MS. A time resolution of 1.5625 ns, per-pixel multihit functionality, simultaneous measurement of TOF and time-over-threshold (TOT) values, and kHz readout rates of the TPX3 extended the m/z detection range of the TPX detector family. The detection of singly charged intact Immunoglobulin M ions of m/z value approaching 1 × 106 Da has been demonstrated. We also discuss the utilization of additional information on impact coordinates and TOT provided by the TPX3 compared to conventional MS detectors for the enhancement of the quality of the mass spectrum in terms of signal-to-noise (S/N) ratio. We show how the reduced dead time and event-based readout in TPX3 compared to the TPX improves the sensitivity of high m/z detection in both low and high mass measurements (m/z range: 757–970,000 Da). We further exploit the imaging capabilities of the TPX3 detector for the spatial and temporal separation of neutral fragments generated by metastable decay at different locations along the field-free flight region by simultaneous application of deflection and retarding fields.

S-2     Figure S1. TOF to m/z conversion curve plotted using 10 samples that encompasses an m/z range from 750 to 970,000 Da. All the TOF data were acquired using an initial acceleration voltage (target plate voltage) of 25 kV.
S-5 Figure S2. Distribution of pixel cluster area (in pixels) of LC 1+ , IgG 3+ , IgG 2+ and IgG 1+ ions, and corresponds to the data shown in Figure 2. A time-bin size of 500 ns was used for the generation of the histogram. µ is the average pixel cluster size that is calculated by , where i=pixel cluster size (in pixels), Ni=number of ion events with pixel cluster size of 'i', and k= maximum pixel cluster size (in pixels).
S-6 Figure S3. TOT distribution of the TPX3 pixels triggered by IgG ions (m/z range: 25-150 kDa), and corresponds to the data shown in Figure 2. A time-bin size of 25 ns was used for the generation of the histogram. S-7 Figure S4. Distribution of pixel cluster area (in pixels) of IgM 1 + -5 + ions, and corresponds to the data shown in Figure 3. A time-bin size of 500 ns was used for the generation of the histogram. µ is the average pixel cluster size that is calculated as previously described ( Figure S2). Figure S5. Distribution of pixel cluster area (in pixels) of the ions generated from Bruker peptide calibration standard II (m/z range: 700-3200 Da), and corresponds to data shown in Figure 4a and b. A time-bin size of 500 ns was used for the generation of the histogram. µ is the average pixel cluster size that is calculated as previously described ( Figure S2). Figure S6. m/z resolved TPX3 images of Trypsinogen 2+ (m/z=11,992), Trypsinogen 1+ (m/z=23,983), Protein A 1+ (m/z=44,613) and BSA 1+ (m/z=66,527) from Bruker protein calibration standard II, and generated from data shown in Figure 4e and f by the accumulation of 1000 laser shots (cpp=counts per pixel).
S-10 Figure S7. Distribution of pixel cluster area (in pixels) of metastable neutrals formed in between the source and deflector (a) and precursor insulin chain B [M+H] 1+ ions (b), and corresponds to data shown in Figure 5b, e and h (Scenario 2). A time-bin size of 500 ns was used for the generation of the histogram. µ is the average pixel cluster size that is calculated as previously described ( Figure  S2).
S-11 Figure S8. TOF spectra correspond to the intact insulin chain B [M+H] 1+ ions (red trace) and metastable neutrals formed prior to the deflector (blue trace). Data was acquired with deflector voltage on and reflectron voltage off (Scenario 2 in Figure  5). Note that the intensities in both cases are normalized to their respective maximum for the better comparison and visualization of the TOF spectra.
S-12 Figure S9. Evolution of the (a) total ion TPX3 images and (b) linear TPX3 detector TOF spectra with an increase in the reflectron voltage (a deflection voltage is also applied). Note that the intensities are normalized to their respective maximum for all the TOF spectra. The data acquisition parameters are listed in Table S1.